Western Blot Analysis

Total protein from cultured HSC-T6 cells and primary HSCs were extracted with RIPA lysis buffer (contained 1% PMSF) (Beyotime, China). Protein of each sample 30–50 μg was separated by SDS-PAGE gel (10%) and then transferred onto PVDF membranes (Millipore Corp, Billerica, MA, United States). The transferred membranes were blocked in 5% skim milk for 3 h in order to break non-specific binding. Subsequently, membranes were incubated with the primary antibody against PTGIS, COL1a1, α-SMA, β-actin, C-myc, Cyclin D1, Bax, Bcl-2, cleaved-casepase 3, DNMT1, DNMT3a and DNMT3b overnight at 4°C, followed by incubation with secondary antibody at room temperature for 1 h. Signals were captured with Bioshine ChemiQ image system. The intensities of each western blotting band were quantified and analyzed by using the Image J software (NIH, Bethesda, MD, United States). The characteristics of antibodies were listed in Table ​Table44.

The characteristics of antibodies.